Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR)
Axel Weber, … , Nina Merete Christiansen, Holger Christiansen
Axel Weber, … , Nina Merete Christiansen, Holger Christiansen
Published January 10, 2011
Citation Information: J Clin Invest. 2011;121(2):545-553. https://doi.org/10.1172/JCI44415.
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Technical Advance Oncology

Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR)

Abstract

Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cell–specific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets for cancer diagnostics. In this study, we mapped ampGRs around the proto-oncogene MYCN of human neuroblastomas using a high-resolution tiling array (HR-TA). Based on the HR-TA data, we were able to precisely describe the telomeric and centromeric borders of the ampGRs and deduce virtual fusion sites of the joined ampGRs (amplicon fusion sites [AFSs]). These AFSs served as blueprints for the subsequent design of AFS bridging PCR assays (AFS-PCRs). Strikingly, these assays were absolutely tumor cell specific and capable of detecting 1 tumor cell in 1 × 106 to 8 × 106 control cells. We successfully proved the in vivo practicability of AFS-PCR by detecting and quantifying the specific AFS DNA of human MYCN-amplified neuroblastomas in the patients’ corresponding peripheral blood and bone marrow samples. Thus, we believe AFS-PCR could become a powerful and nevertheless feasible personalized diagnostic tool applicable to a large number of cancer patients, including children with MYCN-amplified neuroblastomas.

Authors

Axel Weber, Sylvia Taube, Sven Starke, Eckhard Bergmann, Nina Merete Christiansen, Holger Christiansen

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Figure 7

Specific AFS fragments are detectable in samples of different tissue origin of neuroblastoma patients at the time of initial diagnosis and during therapy.

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Specific AFS fragments are detectable in samples of different tissue ori...
(A) Tumor cell contents of the primary tumors (TU) were estimated at 100%. Tumor cell contents of the different probes were quantified according to the 2–ΔΔCt calculation. Each PCR was performed in triplicate. Mean and SD are indicated by points and horizontal bars, respectively. DNA from human placenta tissue served as negative control (Con). AFS-PCR identified a weak BM infiltration for patient TU-20 at the time of diagnosis. This infiltration was not detectable cytologically. Patient TU-21 had considerable BM infiltration in a control aspiration after 6 months of therapy. AFS-PCR of a secondary biopsy sample of the residual tumor (R) showed a 10-fold decrease of tumor cells compared with the initial tumor specimen. Patient TU-23 had BM infiltration and circulating tumor cells in the PB at the time of diagnosis (BM1). The secondary BM aspirations, taken from 2 independent anatomical locations after 1 month of therapy (BM2 and BM3), showed a 10-fold decrease of tumor cells compared with the initial BM sample, giving evidence for a response to therapy. (B) AFS fragments and INHBB control fragments of the RQ-PCR separated in a 3% agarose gel. RQ-PCR had been running for 40 cycles. Asterisks denote lines of the tumor probe loaded with half the PCR product of the other lines.